Abstract

In this study, we used flow a cytometric assay to evaluate plasma membrane integrity and mitochondrial activityin post-thawed sperm that was supplemented with ginsenoside-Rg1. Varying concentrations of ginsenoside-Rg1 (0, 25,50 and 100 μM/ml) were used in the extender during cryopreservation to protect the DNA of thawed sperm, therebyincreasing the viability and motility rate as evaluated using a computer-assisted sperm analysis (CASA) method. Theresults derived from CASA were used to compare the fresh, control, and ginsenoside-Rg1 groups. Sperm motility andthe number of progressively motile sperm were significantly (p<0.05) higher in the 50 μM/ml ginsenoside-Rg1 group(61.0±4.65%) than in the control (46.6±7.02%), 25 μM/ml (46.2±4.76%), and 100 μM/ml ginsenoside-Rg1 (52.0±1.90%) groups. However, the velocity distribution of post-thawed sperm did not differ significantly. Membraneintegrity and MMP staining as revealed using flow cytometry were significantly (p<0.05) higher (91.6±0.82%) in the50 μM/ml ginsenoside-Rg1 group than in the other groups.Here, we report that ginsenoside-Rg1 affects the motility and viability of boar spermatozoa. Moreover, ginsenoside-Rg1 can be used as a protective additive for the suppression of intracellular mitochondrial oxidative stress causedby cryopreservation.

Recently, freezing of boar sperm with optimal extenders, sugars (Hu et al., 2009; Park et al., 2012), and antioxidants (Pena et al., 2003) has improved motility and viability, which can help to resist the formation of intracellular ice crystals and impairment of the sperm plasma membrane. Ginsenoside is a plant-derived glycoside that bears a different chemical structure compared to common plant glycosides; it has well-known antitumour, anti-oxidant and anti-aging effects (Shen et al., 2007; Kong et al., 2010; Huang et al., 2012; Korivi et al., 2012). Ginsenoside-Rg1 is the primary ingredient of Panax ginseng, which a known neuroprotective drug that has been studied for its promotion of DNA synthesis (Zhang et al., 2012a; Zhang et al., 2012b; Wu et al., 2013; Zou et al., 2013). When boar sperm is cryopreserved for long-term storage, reactive oxygen species (ROS) appear within the mitochondria and damage the DNA. Therefore, ROS levels should be monitored because the sperm are not useful if the cells’ mitochondria are damaged and fail to function when improperly thawed. The objective of the present study was to evaluate the effects of ginsenoside-Rg1 on the motility, mitochondrial activity and mitochondrial membrane potential of post-thawed miniature pig sperm. Our experiment compared the effects of ginsenoside-Rg1 at final concentrations of 0, 25, 50, and 100 μM/ml.

2. Ejaculate Collection and Processing

The semen samples were obtained from two miniature pigs in Kangwon National University using the gloved-hand technique and were delivered to our laboratory in Incheon, where they were maintained at 17℃. We used the ejaculates that were >85% motile as well as samples that were >65% progressively motile. After collection, the semen was diluted with modified Modena B extender buffer (mMB, 6 g glucose, 0.45 g EDTA, 1.38 g sodium citrate, 0.2 g sodium bicarbonate, 1 g Tris base, 0.5 g citric acid, 0.01 g cysteine, 0.8 g BSA, and 0.06 g kanamycin sulfate; pH 7).

3. Boar Sperm Cryopreservation

The collected semen was diluted with mMB extender (semen: extender ratio=1:3) and was centrifuged at 400×g for 10 min at 17℃. For the analyses, the samples were washed 3 times with mMB extender. The supernatant was discarded, and an aliquot was adjusted to an optimal concentration and diluted in lactose-egg yolk solution (LEY extender). The purified sperm pellet was suspended in cooling extender (LEY solution: 80% v/v lactose solution [310 mM] and 20% v/v egg yolk with 100 μg/ml kanamycin sulfate) at a density of approximately 1×109 sperm/ml. The sperm suspension was subjected to a gradual cooling stage of 1℃ every 5 min in icewater. Freezing extender (LEYGO solution: 89.5%v/v LEY extender; 9%v/v glycerol; 1.5%v/v OrvusESPaste [OEP]; 100 mM trehalose; and 0, 25, 50, 100 μM/ml ginsenoside-Rg1) was added to the suspensions when a temperature of 4℃ was reached. The sperm suspensions were loaded into 0.25 ml straws and stored in liquid nitrogen.

4. Sperm Evaluation

1) Post-thaw Sperm Motility & Velocity Distribution

Sperm motility was evaluated using a computer-assisted sperm analysis (CASA) system (Hamilton Thorne, Inc., Beverly, MA, USA). The cryopreserved sperm samples were incubated in a 50℃ water bath for 15 sec, and the post-thawed sperm was transferred to 15 ml tubes and diluted with 10 ml mMB extender that was pre-warmed to 37℃. After dilution, the sperm cells were centrifuged at 300×g for 10 min at room temperature. The sperm concentrations were adjusted to 5×107 sperm /ml with mMB. For the analysis, 3 μl of the sperm sample was placed in a counting chamber (20 μm depth) and observed using a microscope; at least 10 predetermined fields were analysed. CASA evaluation yielded the angular path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), the amplitude of lateral displacement of sperm head (ALH), beat cross frequency (BCF) of the sperm head, straightness (STR) and linearity (LIN) of the sperm. The velocity distribution and quality of fresh, control and ginsenoside-Rg1 treated sperm quality were determined using a CASA system.

5. Mitochondria Activity

Post-thawed sperm was washed with D-PBS (0.5 mg/ml phosphate-buffered saline, PBS) and incubated for 10 min at 37℃. Following incubation, the supernatant was removed by centrifugation (500×g, 5 min), and the sperm pellets were resuspended in 1 ml PBS. Ten microliters of the suspension were placed on microscopic slides using coverslips (22 mm×22 mm) and examined at 400×magnification under epifluorescence microscopy (Axiovert 200, Zeiss, Germany). The scope was equipped with excitation/barrier filter of 490/515 nm for R123 (blue excitation), an excitation/barrier filter of 545/590 nm for PI (green excitation) and a digital camera (Canon EOS 550Ds, Tokyo, Japan). Sperm cells displaying only green fluorescence at the mid-piece region were considered to be viable spermatozoa with functional mitochondria. For each aliquot, approximately 200 sperm cells were classified as spermatozoa with viable or non-viable mitochondria.

The sperm were categorised according to the concentration of ginsenoside-Rg1 and underwent fluorescent staining. Propidium iodide (PI), which is the most popular viability stain, can be excited at 488 nm and can enter cells with a broken plasma membrane, emitting red fluorescence at 636 nm when bound to nucleic acid. The Mitochondrial Staining Kit (MitoProbe™ JC-1 Assay Kit, Molecular probe, Eugene, OR) consists of the probe JC-1 that is freely permeable to cells and can undergo reversible transformation from a monomer to an aggregate form (Jagg). This probe fluoresces at 590 nm in response to 488 nm excitation after binding to membranes having an MMP between 80 and 100 mV. Two fluorescent dyes were used to stain sperm for measuring sperm function. Aliquots of 500 μl of sperm sample (1×106 sperm/ml) were mixed with both JC-1 and PI to final concentrations of 0.3 μM and 9.6 μM, respectively.

7. Flow Cytometric Analysis

Flow cytometric analysis was performed using the FC 500 series (Beckman Coulter Inc., Miami, FL, USA). FL2 (JC-1) signals were detected with a 488 nm band pass filter, and FL3 (PI) signals were detected using a 620 nm band pass filter. The flow cytometric data were analysed using the CXP program (Beckman Coulter Inc.). The sperm suspensions stained with fluorescent dye were run through the flow cytometer and a gate was established based on the log forward-scatter (FSC) and side-scatter (SSC) light properties, and the compensation of each probe was determined. To precisely set the region on the sperm and to avoid debris or fragments of cells, two combinations of the two fluorescent dyes were used (JC-1 to identify viable mitochondria, and PI to identify nonviable cells or cells with damaged membranes).

8. Statistical Analysis

The data were processed using the R statistical program (R Development Core Team). The data were expressed as the means±SD and analysed using ANOVA and Tukey statistical tests to determine the limit of detection for the viability and DNA damage after cryopreservation. Differences were considered significant at p<0.05.

RESULT

1. Motility and Velocity Distribution

Sperm motility was measured using a CASA system (Table 1). Sperm motility was improved by the addition of 50 μM/ml ginsenoside-Rg1. The percentage of both motility and progressive motility were significantly higher for the sperm that were cryopreserved in 50 μM/ml ginsenoside-Rg1 group (61.0±4.65% and 34.5±2.07%, respectively) than in the control (46.6±7.02% and 22.0±3.39%, respectively) 25 μM/ml (46.2±4.76% and 24.4±3.65%, respectively), and 100 μM/ml ginsenoside-Rg1 groups (52.0±1.90% and 22.7±1.37, respectively). The majority of the motion parameters analysed (VAP, VSL, VCL, ALH, and LIN) slightly increased with the addition of ginsenoside-Rg1 compared to the control group, but BCF and STR did not differ significantly. However, the velocity distribution of postthawed sperm was not significantly different between the 25, 50, and 100 μM/ml ginsenoside-Rg1-supplemented groups (Table 2). The percentages of rapid sperm that were detected with 25, 50, and 100 μM/ml ginsenoside-Rg1 were 65.8±2.17%, 65.7±3.39%, and 69.8±3.56%, respectively. Different superscripts between rows represent significant differences (p<0.05).

Table 2. The velocity distribution of the boar sperm quality of fresh, control and ginsenoside-Rg1 treated groups

2. Mitochondria Activity

Mitochondrial activity was determined using a Rhodamine 123 probe. Three hundred sperm were counted under a fluorescence microscope. Sperm mitochondria that fluoresced bright green at the mid-piece region were considered to be positive for mitochondrial activity (Fig. 2A). However, the mitochondrial activity of post-thawed sperm did not differ significantly among the 25, 50, and 100 μM/ml ginsenoside-Rg1-supplemented groups (Fig. 2B). The percentages of rapid sperm detected were 74%, 80%, and 76%, respectively. Different superscripts between rows indicate significant differences (p<0.05).

Fig. 2. Mitochondrial activity was determined by a Rhodamine 123 probe. Photomicrograph of fluorescently stained boar spermatozoa with Rhodamine 123 (A). One hundred sperm were counted under a fluorescence microscope. Sperm mitochondria that were visible as by bright green fluorescence in the mid-piece region were considered to be positive for mitochondrial activity (B).

The SYBR/PI analysis revealed the membrane integrity of the thawed boar sperm (Fig. 3). The percentage of live spermatozoa was higher for the 50 μM/ml ginsenoside-Rg1-supplemented group than for the sperm cryopreserved with ginsenoside-Rg1 at final concentrations of 0, 25, and 100 μM/ml (p<0.05). Nevertheless, the analyses of all of the parameters demonstrated a difference between the control and experimental groups (p<0.05).

Sperm membrane integrity of frozen-thawed boar sperm was determined using JC-1 with flow cytometry (Table 3 and Fig. 4). The numbers of intact mitochondria were higher when the extender was supplemented with 50 μM/ml ginsenoside-Rg1 (91.9±0.82%) than when it was supplemented with 0, 25, or 100 μM/ml ginsenoside-Rg1 (79.9±3.69%, 69.5±4.57%, and 23.00±1.68%, respectively) (p<0.05). The numbers of monomer (disrupted) mitochondria were higher in 0, 25, 100 μM/ml ginsenoside-Rg1 group (16.2±1.91%, 18.1±1.27%, and 32.3± 6.02 %, respectively) than in the 50 μM/ml ginsenoside-Rg1 group (4.2±1.55%). The analysis of all of the parameters indicated that there was a difference between the four sets of samples (p<0.05).

Table 3. The mean values of the fluorescence for Control and Ginsenoside-Rg1 treated groups. The parameters shown indicate aggregates (intact) and monomers (disrupted) of mitochondria.

DISCUSSION

It is known from experiments with boar sperm that several sugars and antioxidants can be effective cryoprotectants. In previous studies, the effects of various antioxidants, which prevented oxidative stress on the sperm membrane, have been investigated in avian, boar, bull, ram, equine, and human sperm (Pena et al., 2003). Among those antioxidants, vitamin E may serve as an effective protectant against oxidative damage during the freezing process, which can affect sperm motility.

In the 1970s, Polge et al. utilised glucose and egg yolk for boar sperm freezing. It has also been of interest to develop new and safe cryoprotectants to replace glycerol, which has been reported to have cytotoxic effects (He et al., 2004; Hwang et al., 2009; Park et al., 2012; Macias Garcia et al., 2012). For a long time, Ginseng has been used as an herbal medicine due to its effects on decreasing high blood pressure, enhancing metabolism, and increasing immune function (Han et al., 2006). Ginsenoside-Rg1 is the primary ingredients of Panax ginseng, which is known to prevent lipopolysaccharide-stimulated cytokine production, sepsis and inflammation (Shen et al., 2007; Kong et al., 2010; Huang et al., 2012; Korivi et al., 2012; Zou et al., 2013). Furthermore, ginsenoside-Rg1 has been reported to have anti-apoptotic and antioxidants properties as free radical scavenger (Zou et al., 2013). According to the report by Zhang et al. ginsenoside Re has a beneficial effect on human sperm capacitation and acrosome reaction, and this effect has been implicated in the NO/cGMP/PKG pathway.

In our present study, we evaluated the motility, mitochondrial activity, and mitochondrial membrane potential of postthawed sperm treated with 0, 25, 50 and 100 μM/ml ginsenoside-Rg1 by CASA and flow cytometry. Using flow cytometric analysis of fresh and frozen-thawed (FT) sperm can be valuable due to the accuracy of the approach; specifically, this type of analysis can detect minute differences between samples with statistical reliability and high repeatability (Garner et al., 1997; Martinez-Pastor et al., 2004; Pena et al., 2009; Partyka et al., 2010).

Sperm motility considered ATP consuming process that is an important parameter affected by cryopreservation procedures (Watson, 1995) and a strong predictor of the successful IVF. Mitochondria activity could have decreased for sperm motility consequences (Donnelly et al., 1998; Ogier de Baulny et al., 1999). The percentage of post-thawed sperm with 50 μM/ml ginsenoside-Rg1, which was significantly higher than motility and progressive for control, 25, 100 μM/ml ginsenoside-Rg1. This explains the differences have not found significantly correlation between motility of sperm and concentration of ginsenoside-Rg1. There was not concentration dependent pattern. We assumed that high concentration of ginsenoside-Rg1 was toxic on sperm. These results are correlation with Zhanget al.

In the present study, we used ginsenoside-Rg1 to determine whether it affects the boar sperm mitochondria. We suggest that ginsenoside-Rg1 affects not only the mitochondria of boar sperm as shown in Fig. 2 and Table 2, but also sperm motility, progressive motility and other parameters (Fig. 1 and Table 1). The study also demonstrates that 50 μM/ml ginsenoside-Rg1 is an effective concentration for the cryopreservation of miniature porcine sperm.

Fig. 1. Motility of the boar sperm quality analysis of fresh, control and various concentration of ginsenoside-Rg1. Results are expressed as means±SEM of a total number of analyzed boar sperm of 5,645 (fresh semen), 5,322 (control), 5,051 (25 μM/ml), 7,664 (50 μM/ml), and 6,770 (100 μM/ml). Motility means sperm’s ability to move spontaneously and actively, consuming energy in the process, progressive means sperm’s ability to swim fast in straight line, VAP means the mean velocity of the sperm head along its average trajectory, VSL means the mean path velocity of the sperm head along a straight line from its first to its last position, VCL means the mean path velocity of the sperm head along its trajectory, ALH means the mean value of the extreme side-to side movement of the sperm head in each beat, BCF means the frequency with which the sperm trajectory crosses the average path trajectory cycle, STR means VSL/VAP×100, LIN means VSL/VCL×100.

We anticipate that our study will provide basic information regarding the study of viability and mitochondria of cryopreserved sperm; in addition, if ginsenoside-Rg1 is added to fresh semen when frozen, the viability coefficient of sperm should increase.

ACKNOWLEDGMENTS

The authors also appreciate the sperm sample from Choonkeun Park in the Kangwon National University.